Naturally occurring or endogeneous estrogens constitute one class of steroid sex hormones which are produced in the ovaries and other tissues in the body and which stimulate the growth and development of the secondary sex characteristics in female animals. The naturally occurring estrogens are estrone (also known as E.sub.1), estradiol-17B (also known as E.sub.2), and estriol (also known as E.sub.3) The secretion of such estrogens controls in major part the normal sexual cycle in humans and animals which appears characteristically as the changes of estrus, a period of mating activity marked by intense sexual urge (sexual heat).
Historically, it has been demonstrated that removal of both ovaries from an adult normal female mammal abolishes estrus, that is the period of intense sexual urge and mating activity; equally important, it was found that entering into the state of estrus is accompanied and characterized by visually identifiable and distinct changes in the cell structure of the cells lining the vagina. At the height of estrus, the cell lining acquires a unique, cornified character easily distinguishable from those cells present when the animal is resting, in a non-estrus condition, in an immature animal which does not display full sexual activity of an adult, or has been castrated. On this historical and technical basis, the naturally occurring estrogens were first isolated and purified. Concomittantly, the term "estrogen" is defined only in operational form as--any substance able to induce estrus in a living animal. In consequence, all other pharmacologically active compositions, steroidal and nonsteroidal which were subsequently synthesized and which were demonstrably able to induce estrus or to mimic the induction of estrus have been conventionally classified as "estrogenic" regardless of their chemical structure or their mechanism of action.
The value of naturally occuring estrogens and synthetic compositions demonstrating "estrogenic" activity has been in their medical and therapeutic uses, many of which continue to be the subject of considerable controversy. A traditional listing of the therapeutic applications for estrogens alone or in combination with other active agents includes: oral contraception; relief for the symptoms of menopause; prevention of threatened or habitual abortion; relief of dysmenorrhea; relief of dysfunctional uterine bleeding; an aid in ovarian development; treatment of acne; diminution of excessive growth of body hair in women (hirsutism); the prevention of heart attacks; treatment of osteoporosis; an alternative to surgery for metastatic breast cancer; treatment of prostatic carcinoma; and suppression of post-partum lactation [Goodman and Gilman, The Pharmacological Basis Of Therapeutics (Seventh Edition), Macmillan Publishing Company, 1985, pages 1421-1423]. Accordingly, there has been increasing interest in finding newly synthesized compositions and new uses for previously known compounds which are demonstrably estrogenic, that is, able to induce estrus. Probably the best known examples are: ethinyl estradiol (Estinyl); 3-methyl-ethinyl estradiol (Mestranol); and diethylstilbestrol (DES); methallenestril (Vallestril); and doisynoestrol (Fenocylin).
Despite the apparent wide interest in compositions said to be estrogenic, surprisingly few assay methods are available to detect and identify those compositions which have the pharmacological ability to initiate estrus. To date, only three are recognized: the cornification of vaginal cells in the spayed rodent; the change in weight of excised wet uterine tissue; and the ability of the composition to bind competitively with radiolabeled estradiol in cytosol preparations, and homogenates of immature uterine cells. Each of these will be briefly summarized.
The cornification of vaginal cells is the classical methodology by which the naturally occurring endogenous estrogens were discovered. The assay methodology has remained substantially unaltered since its first use [Allen and Doisy, J. Am. Med. Assoc. 81:819 (1923)]. The assay is based upon the ability of a substance to produce the typical estrus response in-vivo when injected into castrated rats or mice. A positive reaction is characterized by the distinct change in the structure of the cells lining the vagina in which the cells acquire a unique, cornified appearance easily distinguishable from cells in the resting state. Microscopic examination of vaginal smears provides empirical evaluation of the estrus condition of the living animal. Traditionally, the activity of a substance under test is expressed in terms of mouse or rat units. A mouse unit is the quantity of endogenous estrogen that just suffices to produce estrus in a castrated animal. Typically, the mouse unit varies from about 0.04-0.1 microgram (hereinafter "ug") of pure estrone; the international unit of activity has been set as 0.1 ug of estrone.
The uterine wet weight assay identifies substances which induce estrus by measuring the increase in the mass weight of uterine tissues, the increase in weight being directly attributable to the interaction between those substances and the secondary sex organs. The test utilizes immature and/or adult ovariectomized mice and rats which are administered a test substance subcutaneously using an inert fluid such as corn oil. Groups of six to ten animals are typically injected daily for three days with the putative estrogenic substance and sacrificed on the fourth day. The uteri are removed, dissected free of extraneous tissue and the fluid contents expelled. Each excised uterine tissue is then blotted and the wet weight determined individually. The increased wet weight of the uterine tissue in comparison to those from control animals receiving only the inert fluid identifies a potent estrogenic composition [Astwood, Anat. Rec. 70:5 (1938); Kay et al., Biochem. Biophys. Acta. 261:475 (1972); Black et al., Life Sciences 26:1453-1458 (1980)].
The competitive binding assay is based on the existence of a specific estrogen receptor (hereinafter "ER") on the cell or tissue extract to which all putative estrogenic compositions will specifically bind in some measurable degree. The binding of the substance to the ER site is believed by many investigators to be responsible for initiating both protein synthesis and cell proliferation in those cells and tissues having demonstratable ER sites. Although initially described as an in-vivo technique [Jensen and Jacobson, Rec. Prog. Horm. Res. 18:387-414 (1962)] the preferred technique is an in-vitro methodology in which binding of the estrogenic substance occurs in a homogenate of rat uterine tissue which has been prepared as a cytosol, the supernatant fraction containing the soluble proteins after sedimentation at 105,000.times.gravity for one hour [Noteboom and Gorski, Arch. Biochem. Biophys. 111:559-568 (1965); Talwar et al., Proc. Natl. Acad. Sci. U.S.A. 52:1059-1066 (1964)]. This supernatant fraction, the cytosol, is typically combined with the test substance for 30 minutes at 4.degree. C. in a test tube. Controls are prepared using inert fluid carriers and non-specific binding is determined in parallel preferrably using a synthetic estrogen such as DES. Subsequently, radiolabeled [.sup.3 H] estradiol is added to all samples at a predetermined concentration. The incubation is continued after which a suspension of dextran-coated charcoal in a suitable buffer is added to all the samples and allowed to react for approximately 20 minutes' duration. Each control and test sample is then centrifuged to yield a discardable precipitant and a supernatant whose radionuclide content is measured by liquid scintillation counting. [Jordan et al., J. Endocr. 75:305-316 (1977)].
It is noteworthy that all the presently accepted assay techniques, whether in-vivo or in-vitro, are based upon a single common mechanism of action for all estrogens as a class (including endogeneous estrogens, synthetically prepared estrogens and other substances demonstrating estrus inducing activity). The theory accepted almost exclusively is the existence of putative intracellular receptor proteins for the estrogenic substance in the cells of estrogen responsive tissues such as the vaginal lining, uterine tissue, the female breast, the pituitary, and the hypothalamus. Estrogens and estrogenic compositions bind with high affinity to the intracellular receptor protein, termed the estrogen receptor or "ER" site. After binding the formed estrogen-protein complex is said to be converted into a species that is physically translocated to the nucleus of the cell where further binding of the estrogen to the genetic material occurs. Some recent publications have confirmed the data reported previously [Mester et al., Exp. Cell Res. 81:447-452 (1973)] suggesting that estrogens bind directly to unoccupied nuclear ER sites avoiding the translocation step [King and Greene, Nature 307:745-747 (1984); Welshons et al. Nature 307:747-749 (1984)]. Subsequent to these events, a general increase cell proliferation is observed. [Gorski and Gannon, Annu. Rev. Physiol. 38:425-450 (1976); Gorski et al., Recent Prog. Horm. Res. 24:45-72 (1968); Jordan, Pharmacological Reviews 36:245-276 (1984)].
It is ironic that the effort to procure new estrogenic compositions and to obtain evidence which reinforces the hypothesis that a single ER site for estrogen is present in estrogen sensitive cells and tissues has led directly to the recognition and isolation of several different kinds of compounds that in fact inhibit or neutralize the action of endogenous estrogens. These have been termed "antiestrogens". Historically, the weakly estrogenic compound chlorotrianisene, unlike most estrogens, was observed not to cause enlargement of the pituitary when given to rats in large doses. Estrogens normally cause pronounced enlargement of the pituitary, but when chlorotrianisene was given concurrently with estradiol, the effect was greatly reduced [Segal and Thompson, Proc. Soc. Exp. Biol. Med. 91:623-625 (1956)]. Subsequently, the related non-estrogenic compound, ethanoxytriphenol, was found to be strikingly antiestrogenic. It inhibited the activity of endogeneous estrogen as well as of synthetic estrogens such as DES and chlorotrianisene. Subsequently, a large number of other antiestrogens have been identified and classified according to their chemical structure into two different classes: steroidal antiestrogens such as RU16117 which dissociates rapidly from the ER site and RU39411 which forms a more stable complex with the ER protein; and non-steroidal antiestrogens including the triphenylethylenes such as tamoxifen and its various derivatives, enclomiphene, clomiphene, nafoxidine, LY117018 and Keoxifene [Jordan, V., Pharmacological Reviews 36:245-276 (1984); Jordan et al., Cancer Treat. Rep. 64:745-759 (1980); Heel et al., Drugs 16:1 (1978); Legha et al., Ann. Intern. Med. 88:69-77 (1978)].
An "antiestrogen" is usually defined as a compound that will inhibit the vaginal cornification produced by estradiol in ovarectomized rats and/or will inhibit the increase in uterine weight produced by estradiol in immature rats. The existence of a specific antiestrogenic binding site in addition to the traditionally accepted ER binding site on specific cells and tissues has been an area of intense controversy and study. Solely to test this theory, two in-vitro test systems have been developed: the growth of MCF-breast cancer cells - to specifically determine the effects of tamoxifen and its metabolites on cell proliferation [Coezy et al., Cancer Res. 42:317-323 (1982); Reddel et al., Cancer Res. 43:4618-4624 (1983)] and estrogen-stimulated prolactin synthesis by primary cell cultures of immature rat pituitary glands--for the ability of antiestrogens to inhibit prolactin synthesis [Lieberman et al., J. Biol. Chem. 258:4734-4740 (1983); Lieberman et al., Proc. Natl. Acad. Sci. U.S.A. 75:5946-5949 (1978)].
The ability to identify and to distinguish between estrogenic compounds and antiestrogens is recognised as critical by physicians and pharmacologists. Naturally occurring endogenous estrogens are able to induce a variety of multiple changes in their target organs: increased cellular proliferation or hyperplasis; induction of specific protein synthesis; and an increased cellular mass or hypertrophy. From the viewpoint of clinicians and human disease, the hyperplastic effects are the most important because it is central to the problem of hormone-sensitive cancers and their control. Estrogen antagonists thus are of primary interest. From the viewpoint of pharmacologists interested in developing new drugs useful for the treatment of human diseases and specific pathological conditions, it is most important to procure compounds with some demonstrable estrogen-like function but which are devoid of proliferative side-effects. Exemplifying this latter view, osteoporosis, a disease in which bone becomes increasingly more fragile, is greatly ameliorated by the use of fully active estrogens; however, due to the recognized increased risk of cancer in patients chronically treated with active estrogens, it is not clinically advisable to treat osteoporosis with fully active estrogens. Accordingly estrogen agonists are the primary interest and focus.
The difficulties of identifying and evaluating estrogen agonistic compositions and estrogen antagonistic compounds are recognized. In-vivo methods to detect and identify such compositions have been difficult to perform in a consistent manner and are open to independent biological variations which cannot be entirely controlled or eliminated. Equally recognized as a continuing problem are the mechanisms of action for estrogenic and antiestrogen compositions--the evidence for which is often conflicting, extremely complex, and frequently inconsistent. Each class of composition has been shown to have different properties in estrogen target tissues and to induce radically different pharmacological reactions among alternate species of test animals. The best example of the latter is the number of estrogenic and antiestrogenic compositions which have been demonstrated to be active in rats and mice but which have subsequently been found to be inactive or poorly effective in clinical trials using human subjects. Underlying and inherent in each of these problems and difficulties, is the seemingly widely accepted requirement to conform to the generally accepted theory of intracellular ER sites on the target cells and tissues before any in-vivo or in-vitro assay methodology may be deemed effective. As can be readily appreciated, there is a continuing need for novel, reliable, accurate, and sensitive assay protocols which will identify those pharmacological substances which are demonstrably active as estrogens from those which are inhibitors or neutralizers of estrogens and from those substances which are completely quiescent with respect to estrogens.